Combination therapy for treating protein deficiency disorders

ABSTRACT

This application provides methods of improving protein replacement therapy by combining protein replacement therapy with active site-specific chaperones (ASSC) to increase the stability and efficiency of the protein being administered. The application further provides stable compositions comprising the purified protein and an ASSC, and methods of treatment by administering the compositions.

This application is a divisional of U.S. application Ser. No.11/317,404, filed on Dec. 23, 2005 (pending), which is a continuation ofU.S. application Ser. No. 10/771,236, filed on Feb. 2, 2004 (abandoned),which claims priority benefit from U.S. Provisional Application Ser. No.60/444,136, filed Jan. 31, 2003 (abandoned), each of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This application provides methods of improving protein replacementtherapy by combining protein replacement therapy with activesite-specific chaperones (ASSC) to increase the stability and efficiencyof the protein being administered. The application further providescompositions comprising the purified protein and an ASSC.

BACKGROUND Protein Deficiency

Proteins are synthesized intracellularly according to the genomicnucleotide sequence of a particular gene through transcription,translation, and other processes. Protein deficiency can be caused by amutation in the coding gene, which results in (i) non-synthesis of theprotein; (ii) synthesis of the protein which lacks biological activity;or (iii) synthesis of the protein containing normal or partialbiological activity, but which cannot be appropriately processed toreach the native compartment of the protein. Protein deficiencydisorders that result from genetic mutations are also referred to asgenetic disorders.

In addition to protein deficiencies resulting from genetic mutations,some protein deficiencies arise due to a disease, or as a side effect ofa treatment for a disease (e.g., chemotherapy) or as a result ofnutritional insufficiency.

Current Therapies.

There are numerous disorders resulting from protein deficiencies, someof which result from mutated, misfolded proteins (conformationaldisorders-see infra). One current therapy for treating proteindeficiencies is protein replacement therapy, which typically involvesintravenous, subcutaneous or intramuscular infusion of a purified formof the corresponding wild-type protein, or implantation of the proteinin a bio-erodable solid form for extended-release. One of the maincomplications with protein replacement therapy is attainment andmaintenance of therapeutically effective amounts of protein due to rapiddegradation of the infused protein. The current approach to overcomethis problem is to perform numerous costly high dose infusions.

Protein replacement therapy has several additional caveats, such asdifficulties with large-scale generation, purification and storage ofproperly folded protein, obtaining glycosylated native protein,generation of an anti-protein immune response, and inability of proteinto cross the blood-brain barrier in diseases having significant centralnervous system involvement.

Gene therapy using recombinant vectors containing nucleic acid sequencesthat encode a functional protein, or genetically modified human cellsthat express a functional protein, is also being used to treat proteindeficiencies and other disorders that benefit from protein replacement.Although promising, this approach is also limited by technicaldifficulties such as the inability of vectors to infect or transducedividing cells, low expression of the target gene, and regulation ofexpression once the gene is delivered.

A third, relatively recent approach to treating protein deficienciesinvolves the use of small molecule inhibitors to reduce the naturalsubstrate of deficient enzyme proteins, thereby ameliorating thepathology. This “substrate deprivation” approach has been specificallydescribed for a class of about 40 related enzyme disorders calledlysosomal storage disorders or glycosphingolipid storage disorders.These heritable disorders are characterized by deficiencies in lysosomalenzymes that catalyze the breakdown of glycolipids in cells, resultingin an abnormal accumulation of lipids which disrupts cellular function.The small molecule inhibitors proposed for use as therapy are specificfor inhibiting the enzymes involved in synthesis of glycolipids,reducing the amount of cellular glycolipid that needs to be broken downby the deficient enzyme. This approach is also limited in thatglycolipids are necessary for biological function, and excessdeprivation may cause adverse effects. Specifically, glycolipids areused by the brain to send signals from the gangliosides of neurons toanother. If there are too few or too many glycolipids, the ability ofthe neuron to send signals is impeded.

A fourth approach, discussed below as specific chaperone strategy,rescues mutant proteins from degradation in the endoplasmic reticulum.

Protein Processing in the Endoplasmic Reticulum

Proteins are synthesized in the cytoplasm, and the newly synthesizedproteins are secreted into the lumen of the endoplasmic reticulum (ER)in a largely unfolded state. In general, protein folding is governed bythe principle of self assembly. Newly synthesized polypeptides fold intotheir native conformation based on their amino acid sequences (Anfinsenet al., Adv. Protein Chem. 1975; 29:205-300). In vivo, protein foldingis complicated, because the combination of ambient temperature and highprotein concentration stimulates the process of aggregation, in whichamino acids normally buried in the hydrophobic core interact with theirneighbors non-specifically. To avoid this problem, protein folding isusually facilitated by a special group of proteins called molecularchaperones which prevent nascent polypeptide chains from aggregating,and bind to unfolded protein such that the protein refolds in the nativeconformation (Hartl, Nature 1996; 381:571-580).

Molecular chaperones are present in virtually all types of cells and inmost cellular compartments. Some are involved in the transport ofproteins and permit cells to survive under stresses such as heat shockand glucose starvation (Gething et al., Nature 1992; 355:33-45; Caplan,Trends Cell. Biol. 1999; 9:262-268; Lin et al., Mol. Biol. Cell. 1993;4:109-1119; Bergeron et al., Trends Biochem. Sci. 1994; 19:124-128).Among the molecular chaperones, Bip (immunoglobulin heavy-chain bindingprotein, Grp78) is the best characterized chaperone of the ER (Haas,Curr. Top. Microbiol. Immunol. 1991; 167:71-82). Like other molecularchaperones, Bip interacts with many secretory and membrane proteinswithin the ER throughout their maturation, although the interaction isnormally weak and short-lived when the folding proceeds smoothly. Oncethe native protein conformation is achieved, the molecular chaperone nolonger interacts with the protein. Bip binding to a protein that failsto fold, assemble or be properly glycosylated, becomes stable, and leadsto degradation of the protein through the ER-associated degradationpathway. This process serves as a “quality control” system in the ER,ensuring that only those properly folded and assembled proteins aretransported out of the ER for further maturation, and improperly foldedproteins are retained for subsequent degradation (Hurtley et al., Annu.Rev. Cell. Biol. 1989; 5:277-307).

Certain DNA mutations result in amino acid substitutions that furtherimpede, and in many cases preclude, proper folding of the mutantproteins. To correct these misfoldings, investigators have attempted touse various molecules. High concentrations of glycerol,dimethylsulfoxide (DMSO), trimethylamine N-oxide (TMAO), or deuteratedwater have been shown to suppress the degradation pathway and increasethe intracellular trafficking of mutant protein in several diseases(Brown et al., Cell Stress Chaperones 1996; 1:117-125; Burrows et al.,Proc. Natl. Acad. Sci. USA. 2000; 97:1796-801). These compounds areconsidered non-specific chemical chaperones to improve the generalprotein folding, although the mechanism of the function is stillunknown. The high doses of this class of compounds required for efficacymakes them difficult or inappropriate to use clinically, although theyare useful for the biochemical examination of folding defect of aprotein intracellularly. These compounds also lack specificity.

Specific Chaperone Strategy

Previous patents and publications described a therapeutic strategy forrescuing endogenous enzyme proteins, specifically misfolded lysosomalenzymes, from degradation by the ER quality control machinery. Thisstrategy employs small molecule reversible competitive inhibitorsspecific for a defective lysosomal enzyme associated with a particularlysosomal disorder. The strategy is as follows: since the mutant enzymeprotein folds improperly in the ER (Ishii et al., Biochem. Biophys. Res.Comm. 1996; 220: 812-815), the enzyme protein is retarded in the normaltransport pathway (ER→Golgi apparatus→endosome→lysosome) and rapidlydegraded. Therefore, a functional compound which facilitates the correctfolding of a mutant protein will serve as a site-specific chaperone forthe mutant protein to promote the smooth escape from the ER qualitycontrol system. Since some inhibitors of an enzyme are known to occupythe catalytic center of enzyme, resulting in stabilization of itsconformation in vitro. These specific chaperones may be designatedactive site-specific chaperones (ASSC).

The strategy has been specifically demonstrated for enzymes involved inthe lysosomal storage disorders in U.S. Pat. Nos. 6,274,597, 6,583,158,6,589,964, and 6,599,919, to Fan et al., and in pending U.S. applicationSer. No. 10/304,396 filed Nov. 26, 2002, which are hereby incorporatedherein by reference in their entirety. For example, a small moleculederivative of galactose, 1-deoxygalactonojirimycin (DGJ), a potentcompetitive inhibitor of the mutant Fabry enzyme α-galactosidase A(α-Gal A), effectively increased in vitro stability of a mutant α-Gal A(R301Q) at neutral pH and enhanced the mutant enzyme activity inlymphoblasts established from Fabry patients with R301Q or Q279Emutations. Furthermore, oral administration of DGJ to transgenic miceoverexpressing a mutant (R301Q) α-Gal A substantially elevated theenzyme activity in major organs (Fan et al., Nature Med. 1999; 5:112-115). Successful rescue of a misfolded protein depends on achievinga concentration of the specific inhibitor in vivo that is lower thannecessary to completely inhibit the enzyme, in contrast to the substratedeprivation approach in which enzyme inhibitory concentrations arerequired.

In addition to the lysosomal storage disorders, a large and diversenumber of diseases are now recognized as conformational diseases thatare caused by adoption of non-native protein conformations, which maylead to retardation of the protein in the ER and ultimate degradation ofthe proteins (Kuznetsov et al., N. Engl. J. Med. 1998; 339:1688-1695;Thomas et al., Trends Biochem. Sci. 1995; 20:456-459; Bychkova et al.,FEBS Lett. 1995; 359:6-8; Brooks, FEBS Lett. 1997; 409:115-120). ASSC'shave been shown to rescue expression of mutant proteins other thanenzymes. For example, small synthetic compounds were found to stabilizethe DNA binding domain of mutant forms of the tumor suppressor proteinp53; thereby allowing the protein to maintain an active conformation(Foster et al.; Science 1999; 286:2507-10). Synthesis of receptors hasbeen shown to be rescued by small molecule receptor antagonists andligands (Morello et al., J. Clin. Invest. 2000; 105: 887-95; Petaja-Repoet al., EMBO J. 2002; 21:1628-37.) Even pharmacological rescue ofmembrane channel proteins and other plasma membrane transporters hasbeen demonstrated using channel-blocking drugs or substrates (Rajamaniet al., Circulation 2002; 105:2830-5; Zhou et al., J. Biol. Chem. 1999;274:31123-26; Loo et al., J. Biol. Chem 1997; 272: 709-12). All of theabove references indicate that ASSC's are capable of specific rescue ofmutant proteins including, but not limited to, enzymes, receptors,membrane channel proteins, and DNA transcription factors.

In addition to mutant proteins, ASSC's have also been shown to stabilizewild-type proteins, resulting in their enhanced production andstability. As one example, it has been demonstrated that a specificASSC, DGJ, is able to increase the amount and activity of wild-typeα-Gal A in COS-7 cells transfected with a vector coding the wild-typeα-Gal A sequence. The ASSC rescues the overexpressed wild-type enzyme,which is otherwise retarded in the ER quality control system, becauseoverexpression and over production of the enzyme in the COS-7 cellsexceeds the capacity of the system and leads to aggregation anddegradation (see U.S. application Ser. No. 10/377,179, filed Feb. 28,2003).

In summary, there is a need in the art for methods of improving thebiological and cost efficiency of protein replacement therapy, such asfor the treatment of protein deficiencies or other disorders wherebyreplacement proteins are administered.

SUMMARY OF THE INVENTION

The present invention provides a method for enhancing the stability of apurified protein, which method comprises contacting the protein in apharmaceutically acceptable carrier with an active site-specificchaperone.

The purified protein can be a recombinant protein, and eitherfull-length or truncated while retaining activity.

The present invention also provides a method of increasing in vitro theshelf-life of a protein by contacting the protein in a pharmaceuticallyacceptable carrier with an active site-specific chaperone.

The protein in the pharmaceutically acceptable carrier can belyophilized or an aqueous solution.

The present invention further provides a method of extending thehalf-life and prolonging the activity in vivo of a purified protein inan individual who has been administered the protein in apharmaceutically acceptable carrier, which method comprises contactingthe protein with an active site-specific chaperone in a pharmaceuticallyacceptable carrier.

The present invention provides a method of treatment for an individualhaving a disorder requiring protein replacement, (e.g., proteindeficiency disorders) comprising administering to the individual apurified replacement protein and an active site-specific chaperone(ASSC) capable of stabilizing the replacement protein.

In one embodiment, the replacement protein is a protein associated witha conformational disorder.

In a preferred embodiment, the conformational disorder is a lysosomalstorage disorder.

In one embodiment, the lysosomal storage disorder is Fabry disease.

In another embodiment, the lysosomal storage disorder is Gaucherdisease.

The invention also provides a method for enhancing the stability of amutant, endogenous protein that is deficient due to defective folding orprocessing in the ER concurrently with protein replacement therapy.Stability and, hence, activity of the endogenous protein will beenhanced concurrently with the increased stability of the administeredreplacement protein that corresponds to the mutant protein.

The invention further provides a method for increasing the production ofrecombinant protein by non-mammalian host cells by contacting the hostcell in a medium comprising an ASSC for the protein.

The invention further provides a composition comprising a purifiedprotein and an ASSC for the purified protein in a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates improved stability of both wild type α-Gal Apurified from culture medium of Sf-9 cells infected with recombinantbaculovirus carrying human wild type α-Gal A cDNA, and mutant α-Gal Acollected as homogenates of hearts of transgenic mice overexpressinghuman mutant (R301Q) α-Gal A, respectively, using a site-specificchaperone 1-deoxygalactonojirimycin (DGJ, 1 μM). The mice were treatedwith 0.5 mM DGJ as drinking water for one week prior to the experiment.The mutant (A) and wild type (B) enzymes were pre-incubated with 0.1 Mcitrate-phosphate buffer (pH 7.0) at 37° C. for the mutant enzyme and42° C. for the wild type enzyme, respectively, in the presence of DGJ ata concentration of 1 μM (∘), 0.1 μM (●), 0.03 μM (♦) or 0 μM (no DGJ;⋄). Enzyme activity is reported relative to the enzyme withoutpre-incubation. DGJ can serve as a stabilizer to prevent thedenaturation/degradation of the mutant and wild type enzymes.

DETAILED DESCRIPTION

The present invention advantageously improves the efficiency of proteinreplacement therapy to treat diseases or disorders by contacting theprotein with an active site-specific chaperone (ASSC). The advantages ofthe invention flow from (a) increased efficiency of protein productionfrom non-mammalian cells; (b) increased stability of the therapeuticprotein, manifested by longer shelf life and better in vivo half lifeand activity; (c) maintenance of protein active site structure duringtranslocations in vivo, including across cell membranes; and (d) rescueof endogenous mutant protein that is misfolded during synthesis andconsequently cleared from the endoplasmic reticulum.

The present invention further provides formulations comprising theprotein and active site-specific chaperone (ASSC) specific for thestabilization of the protein.

The invention is based on the discovery that ASSC's can be used as acombination therapy with protein replacement therapy for the treatmentof genetic and other disorders. ASSC's can be screened and identifiedusing methods known in the art. Once a specific ASSC useful for aparticular disorder is identified, the ASSC can be administered to apatient receiving protein replacement therapy to enhance uptake of thereplacement protein in the appropriate cellular compartment, improvestability of the protein in circulation and, if necessary, duringtransport into the cell. The chaperone can stabilize the protein in itsactive form during manufacture, storage and use in vivo.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

Specific Definitions

The term “protein replacement” refers to the introduction of anon-native, purified protein into an individual having a deficiency insuch protein. The administered protein can be obtained from naturalsources (such as human gammaglobulin for treating RSV or mononucleosis)or by recombinant expression (as described in greater detail below). Theterm also refers to the introduction of a purified protein in anindividual otherwise requiring or benefiting from administration of apurified protein, e.g., suffering from protein insufficiency. Theintroduced protein may be a purified, recombinant protein produced invitro, or protein purified from isolated tissue or fluid, such as, e.g,placenta or animal milk, or from plants.

The term “disorder characterized by a protein deficiency” refers to anydisorder that presents with a pathology caused by absent or insufficientamounts of a protein. This term encompasses protein folding disorders,i.e., conformational disorders, that result in a biologically inactiveprotein product. Protein insufficiency can be involved in infectiousdiseases, immunosuppression, organ failure, glandular problems,radiation illness, nutritional deficiency, poisoning, or otherenvironmental or external insults.

The term “stabilize a proper conformation” refers to the ability of acompound or peptide or other molecule to associate with a wild-typeprotein, or to a mutant protein that can perform its wild-type functionin vitro in, e.g., a formulation, and in vivo, in such a way that thestructure of the wild-type or mutant protein can be maintained as itsnative or proper form. This effect may manifest itself practicallythrough one or more of (i) increased shelf-life of the protein; (ii)higher activity per unit/amount of protein; or (iii) greater in vivoefficacy. It may be observed experimentally through increased yield fromthe BR during expression; greater resistance to unfolding due totemperature increases, or the present of chaotropic agents, and bysimilar means.

As used herein, the term “conformational disorder” or “conformationaldisease” refers to a disorder that is caused by adoption of a proteinconformation that is not normally formed by a wild-type protein in anative condition with normal biological activity, which may lead toretardation and destruction of a protein in the ER. The decreasedprotein level results in a physiological imbalance that manifests itselfas a disease or disorder. In a specific embodiment, the conformationaldisorder is a lysosomal storage disorder.

As used herein, the term “active site” refers to the region of a proteinthat has some specific biological activity. For example, it can be asite that binds a substrate or other binding partner and contributes theamino acid residues that directly participate in the making and breakingof chemical bonds. Active sites in this invention can encompasscatalytic sites of enzymes, antigen biding sites of antibodies, ligandbinding domains of receptors, binding domains of regulators, or receptorbinding domains of secreted proteins. The active sites can alsoencompass transactivation, protein-protein interaction, or DNA bindingdomains of transcription factors and regulators.

As used herein, the term “active site-specific chaperone” refers to anymolecule including a protein, peptide, nucleic acid, carbohydrate, etc.that specifically interacts reversibly with an active site of a proteinand enhances formation of a stable molecular conformation. As usedherein, “active site-specific chaperone” does not include endogenousgeneral chaperones present in the ER of cells such as Bip, calnexin orcalreticulin, or general, non-specific chemical chaperones such asdeuterated water, DMSO, or TMAO.

General Definitions

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 95% pure; morepreferably, at least 97% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art. In a specific embodiment, purified means that thelevel of contaminants is below a level acceptable to regulatoryauthorities for administration to a human or non-human animal.

In preferred embodiments, the terms “about” and “approximately” shallgenerally mean an acceptable degree of error for the quantity measuredgiven the nature or precision of the measurements. Typical, exemplarydegrees of error are within 20 percent (%), preferably within 10%, andmore preferably within 5% of a given value or range of values.Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 10- or 5-fold, and more preferably within2-fold of a given value. Numerical quantities given herein areapproximate unless stated otherwise, meaning that the term “about” or“approximately” can be inferred when not expressly stated.

A “gene” is a sequence of nucleotides which code for a functional “geneproduct”. Generally, a gene product is a functional protein. However, agene product can also be another type of molecule in a cell, such as anRNA (e.g., a tRNA or a rRNA). For the purposes of the present invention,a gene product also refers to an mRNA sequence which may be found in acell.

The term “express” and “expression” means allowing or causing theinformation in a gene or DNA sequence to become, manifest, for exampleproducing RNA (such as rRNA or mRNA) or a protein by activating thecellular functions involved in transcription and translation of acorresponding gene or DNA sequence. A DNA sequence is expressed by acell to form an “expression product” such as an RNA (e.g., a mRNA or arRNA) or a protein. The expression product itself, e.g., the resultingRNA or protein, may also said to be “expressed” by the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequenceinto a host cell so that the host cell will express the introduced geneor sequence to produce a desired substance, in this invention typicallyan RNA coded by the introduced gene or sequence, but also a protein oran enzyme coded by the introduced gene or sequence. The introduced geneor sequence may also be called a “cloned” or “foreign” gene or sequence,may include regulatory or control sequences (e.g., start, stop,promoter, signal, secretion or other sequences used by a cell's geneticmachinery). The gene or sequence may include nonfunctional sequences orsequences with no known function. A host cell that receives andexpresses introduced DNA or RNA has been “transformed” and is a“transformant” or a “clone”. The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells such as Sf9, Hi5 or S2 cells and Baculovirusvectors, and expression systems, and mammalian host cells and vectors.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g., DNA, or any process, mechanism or result of such achange. This includes gene mutations, in which the structure (e.g., DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g., RNA, protein orenzyme) expressed by a modified gene or DNA sequence.

As used herein the term “mutant protein” refers to proteins translatedfrom genes containing genetic mutations that result in altered proteinsequences. In a specific embodiment, such mutations result in theinability of the protein to achieve its native conformation under theconditions normally present in the ER. The failure to achieve thisconformation results in these proteins being degraded, rather than beingtransported through their normal pathway in the protein transport systemto their proper location within the cell. Other mutations can result indecreased activity or more rapid turnover.

A “wild-type gene” refers to a nucleic acid sequences which encodes aprotein capable of having normal biological functional activity in vivo.The wild-type nucleic acid sequence may contain nucleotide changes thatdiffer from the known, published sequence, as long as the changes resultin amino acid substitutions having little or no effect on the biologicalactivity. The term wild-type may also include nucleic acid sequencesengineered to encode a protein capable of increased or enhanced activityrelative to the endogenous or native protein.

A “wild-type protein” refers to any protein encoded by a wild-type genethat is capable of having functional biological activity when expressedor introduced in vive. The term “normal wild-type activity” refers tothe normal physiological function of a protein in a cell. Suchfunctionality can be tested by any means known to establishfunctionality of a protein.

The term “genetically modified” refers to cells that express aparticular gene product following introduction of a nucleic acidcomprising a coding sequence which encodes the gene product, along withregulatory elements that control expression of the coding sequence.Introduction of the nucleic acid may be accomplished by any method knownin the art including gene targeting and homologous recombination. Asused herein, the term also includes cells that have been engineered toexpress or overexpress an endogenous gene or gene product not normallyexpressed by such cell, e.g., by gene activation technology.

The phrase “pharmaceutically acceptable”, whether used in connectionwith the pharmaceutical compositions of the invention, refers tomolecular entities and compositions that are physiologically tolerableand do not typically produce untoward reactions when administered to ahuman. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the compound is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils. Water oraqueous solution saline solutions and aqueous dextrose and glycerolsolutions are preferably employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition.

The terms “therapeutically effective dose” and “effective amount” referto the amount of the compound that is sufficient to result in atherapeutic response. In embodiments where an ASSC and protein areadministered in a complex, the terms “therapeutically effective dose”and “effective amount” may refer to the amount of the complex that issufficient to result in a therapeutic response. A therapeutic responsemay be any response that a user (e.g., a clinician) will recognize as aneffective response to the therapy. Thus, a therapeutic response willgenerally be an amelioration of one or more symptoms of a disease ordisorder.

It should be noted that a concentration of the ASSC that is inhibitoryduring in vitro production, transportation, or storage of the purifiedtherapeutic protein may still constitute an “effective amount” forpurposes of this invention because of dilution (and consequent shift inbinding due to the change in equilibrium), bioavailability andmetabolism of the ASSC upon administration in vivo.

Disorders Characterized by Protein Deficiencies

There currently are about 1100 known inherited disorders characterizedby protein deficiency or loss-of-function in specific tissue. Thesedisorders may be treatable by protein replacement therapy in theory. Themethod of the present invention contemplates co-therapy for proteinscurrently suited for use in protein replacement therapy that isavailable now or will be in the future. In such disorders, certain cellsor all of the cells of an individual lack a sufficient functionalprotein, contain an inactive form of the protein or contain insufficientlevels for biological function.

Further, the list of diseases identified as being conformationaldisorders, caused by mutations that alter protein folding andretardation of the mutant protein in the ER, resulting in proteindeficiency, is increasing. These include cystic fibrosis, α1-antitrypsindeficiency, familial hypercholesterolemia, Fabry disease, Alzheimer'sdisease (Selkoe, Annu. Rev. Neurosci. 1994; 17:489-517), osteogenesisimperfecta (Chessler et al., J. Biol. Chem. 1993; 268:18226-18233),carbohydrate-deficient glycoprotein syndrome (Marquardt et al., Eur. J.Cell. Biol. 1995; 66: 268-273), Maroteaux-Lamy syndrome (Bradford etal., Biochem. J. 1999; 341:193-201), hereditary blindness (Kaushal etal., Biochemistry 1994; 33:6121-8), Glanzmann thrombasthenia (Kato etal., Blood 1992; 79:3212-8), hereditary factor VII deficiency (Arbini etal., Blood 1996; 87:5085-94), oculocutaneous albinism (Halaban et al.,Proc. Natl. Acad. Sci. USA. 2000; 97:5889-94) and protein C deficiency(Katsumi, et al., Blood 1996; 87:4164-75). Recently, one mutation in theX-linked disease adrenoleukodystrophy (ALD), resulted in misfolding ofthe defective peroxisome transporter which could be rescued bylow-temperature cultivation of affected cells (Walter et al., Am J HumGenet 2001; 69:35-48). It is generally accepted that mutations takeplace evenly over the entire sequence of a gene. Therefore, it ispredictable that the phenotype resulting from misfolding of thedeficient protein exists in many other genetic disorders.

Lysosomal Storage Disorders

Many of the inherited protein deficient disorders are enzymedeficiencies. As indicated above, a large class of inherited enzymedisorders involves mutations in lysosomal enzymes and are referred to aslysosomal storage disorders (LSDs). Lysosomal storage disorders are agroup of diseases caused by the accumulation of glycosphingolipids,glycogen, mucopolysaccharides Examples of lysosomal disorders includebut are not limited to Gaucher disease (Beutler et al., The Metabolicand Molecular Bases of Inherited Disease, 8th ed. 2001 Scriver et al.,ed. pp. 3635-3668, McGraw-Hill, New York), GM1-gangliosidosis (id. at pp3775-3810), fucosidosis (The Metabolic and Molecular Bases of InheritedDisease 1995. Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D.,ed pp. 2529-2561, McGraw-Hill, New York), mucopolysaccharidoses (id. atpp 3421-3452), Pompe disease (id. at pp. 3389-3420), Hurler-Scheiedisease (Weismann et al., Science 1970; 169, 72-74), Niemann-Pick A andB diseases, (The Metabolic and Molecular Bases of Inherited Disease 8thed. 2001. Scriver et al. ed., pp 3589-3610, McGraw-Hill, New York), andFabry disease (id. at pp. 3733-3774). A list of LSDs and theirassociated deficient enzymes can be found in Table 1 infra. Two arediscussed specifically below.

Fabry Disease

Fabry disease is an X-linked inborn error of glycosphingolipidmetabolism caused by deficient lysosomal α-galactosidase A (α-Gal A)activity (Desnick et al., The Metabolic and Molecular Bases of InheritedDisease, 8^(th) Edition Scriver et al. ed., pp. 3733-3774, McGraw-Hill,New York 2001; Brady et al., N. Engl. J. Med. 1967; 276, 1163-1167).This enzymatic defect leads to the progressive deposition of neutralglycosphingolipids with α-galactosyl residues, predominantlyglobotriaosylceramide (GL-3), in body fluids and tissue lysosomes. Thefrequency of the disease is estimated to be about 1:40,000 in males, andis reported throughout the world within different ethnic groups. Inclassically affected males, the clinical manifestations includeangiokeratoma, acroparesthesias, hypohidrosis, and characteristiccorneal and lenticular opacities (The Metabolic and Molecular Bases ofInherited Disease, 8^(th) Edition 2001, Scriver et al., ed., pp.3733-3774, McGraw-Hill, New York). The affected male's life expectancyis reduced, and death usually occurs in the fourth or fifth decade as aresult of vascular disease of the heart, brain, and/or kidneys. Incontrast, patients with the milder “cardiac variant” normally have 5-15%of normal α-Gal A activity, and present with left ventricularhypertrophy or a cardiomyopathy. These cardiac variant patients remainessentially asymptomatic when their classically, affected counterpartsare severely compromised. Recently, cardiac variants were found in 11%of adult male patients with unexplained left ventricular hypertrophiccardiomyopathy, suggesting that Fabry disease may be more frequent thanpreviously estimated (Nakao et al., N. Engl. J. Med. 1995; 333:288-293). The α-Gal A gene has been mapped to Xq22, (Bishop et al., Am.J. Hum. Genet. 1985; 37: A144), and the full-length cDNA and entire12-kb genomic sequences encoding α-Gal A have been reported (Calhoun etal., Proc. Natl. Acad. Sci. USA 1985; 82: 7364-7368; Bishop et al.,Proc. Natl. Acad. Sci. USA 1986; 83: 4859-4863; Tsuji et al., Eur. J.Biochem. 1987; 165: 275-280; and Kornreich et al., Nucleic Acids Res.1989; 17: 3301-3302). There is a marked genetic heterogeneity ofmutations that cause Fabry disease (The Metabolic and Molecular Bases ofInherited Disease, 8^(th) Edition 2001, Scriver et al., ed., pp.3733-3774, McGraw-Hill, New York.; Eng et al., Am. J. Hum. Genet. 1993;53: 1186-1197; Eng et al., Mol. Med. 1997; 3: 174-182; and Davies etal., Eur. J. Hum. Genet. 1996; 4: 219-224). To date, a variety ofmissense, nonsense, and splicing mutations, in addition to smalldeletions and insertions, and larger gene rearrangements have beenreported.

Gaucher Disease

Gaucher disease is a deficiency of the lysosomal enzymeβ-glucocerebrosidase that breaks down fatty glucocerebrosides. The fatthen accumulates, mostly in the liver, spleen and bone marrow. Gaucherdisease can result in pain, fatigue, jaundice, bone damage, anemia andeven death. There are three clinical phenotypes of Gaucher disease.Patients with, Type 1 manifest either early in life or in youngadulthood, bruise easily and experience fatigue due to anemia, low bloodplatelets, enlargement of the liver and spleen, weakening of theskeleton, and in some instances have lung and kidney impairment. Thereare no signs of brain involvement. In Type II, early-onset, liver andspleen enlargement occurs by 3 months of age and there is extensivebrain involvement. There is a high mortality rate by age 2. Type III ischaracterized by liver and spleen enlargement and brain seizures. Theβ-glucocerebrosidase gene is located on the human 1q21 chromosome. Itsprotein precursor contains 536 amino acids and its mature protein is 497amino acids long.

Gaucher disease is considerably more common in the descendants of Jewishpeople from Eastern Europe (Ashkenazi), although individuals from anyethnic group may be affected. Among the Ashkenazi Jewish population,Gaucher disease is the most common genetic disorder, with an incidenceof approximately 1 in 450 persons. In the general public, Gaucherdisease affects approximately 1 in 100,000 persons. According to theNational Gaucher Foundation, 2,500 Americans suffer from Gaucherdisease.

Other Enzyme Deficiency Disorders

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most commonX-linked human enzyme deficiency. The G6PD enzyme catalyzes anoxidation/reduction reaction that is essential for the production ofribose, which is an essential component of both DNA and RNA. G6PD alsoinvolved in maintaining adequate levels of NADPH inside the cell. NADPHis a required cofactor in many biosynthetic reactions. Individuals withthis deficiency have clinical symptoms including neonatal jaundice,abdominal and/or back pain, dizziness, headache, dyspnea (irregularbreathing), and palpitations.

In addition to inherited disorders, other enzyme deficiencies arise fromdamage to a tissue or organ resulting from primary or secondarydisorders. For example, damaged pancreatic tissue, or pancreatitis, iscaused by alcoholism results in a deficiency in pancreatic enzymesnecessary for digestion. Pancreatitis is currently being treated usingenzyme replacement therapy.

TABLE 1 Lysosomal Storage Disorders, Associated Defective Enzymes andSmall Molecule Active Site-Specific Chaperones DISORDER DEFICIENT ENZYMEREVERSIBLE CHAPERONE Pompe disease α-Glucosidase 1-deoxynojirimycin(DNJ) α-homonojirimycin castanospermine Gaucher Acid β-Glucosidaseisofagomine disease (glucocerebrosidase) N-dodecyl-DNJ calystegines A₃,B₁, B₂ and C₁ Fabry disease α-Galactosidase A 1-deoxygalactonojirimycin(DGJ) α-allo-homonojirimycin α-galacto-homonojirimycinβ-1-C-butyl-deoxynojirimycin calystegines A₂ and B₂ N-methylcalystegines A₂ and B₂ G_(M1)- Acid β-Galactosidase 4-epi-isofagominegangliosidosis 1-deoxygalactonojirimycin Krabbe diseaseGalactocerebrosidase 4-epi-isofagomine 1-deoxygalactonojirimycin MorquioAcid β-Galactosidase 4-epi-isofagomine disease B1-deoxygalactonojirimycin α-Mannosidosis Acid α-Mannosidase1-deoxymannojirimycin Swainsonine Mannostatin A β-Mannosidosis Acidβ-Mannosidase 2-hydroxy-isofagomine Fucosidosis Acid α-L-fucosidase1-deoxyfuconojirimycin β-homofuconojirimycin 2,5-imino-1,2,5-trideoxy-L-glucitol 2,5-deoxy-2,5-imino-D-fucitol 2,5-imino-1,2,5-trideoxy-D-altritol Sanfilippo α-N- 1,2-dideoxy-2-N-acetamido- disease BAcetylglucosaminidase nojirimycin Schindler α-N-1,2-dideoxy-2-N-acetamido- disease Acetylgalactosaminidasegalactonojirimycin Tay-Sachs β-Hexosaminidase A2-N-acetylamino-isofagomine disease 1,2-dideoxy-2-acetamido- nojirimycinnagstain Sandhoff β-Hexosaminidase B 2-N-acetamido-isofagomine disease1,2-dideoxy-2-acetamido- nojirimycin nagstain Hurler-Scheieα-L-Iduronidase 1-deoxyiduronojirimycin disease 2-carboxy-3,4,5-trideoxypiperidine Sly disease β-Glucuronidase 6-carboxy-isofagomine2-carboxy-3,4,5- trideoxypiperidine Sialidosis Sialidase2,6-dideoxy-2,6, imino-sialic acid Siastatin B Hunter disease Iduronatesulfatase 2,5-anhydromannitol-6- sulphate Niemann-Pick Acidsphingomyelinase desipramine, disease phosphatidylinositol-4,5-diphosphate

Other Disorders Treated Using Protein Replacement

In addition to disorders characterized by protein deficiencies, somedisorders are treated by administration of replacement proteins toenhance or stimulate biological processes. For example, individuals withanemia are administered recombinant erythropoietin (EPOGEN®, PROCRIT®,EPOIETIN®) to stimulate red blood cell production and increase oxygentransportation to tissues. In addition, recombinant interferons such asinterferon alpha 2b (INTRON A®, PEG-INTRON®, or REBETOL®), andinterferon beta 1a (AVONEX®, BETASERON®) are administered to treathepatitis B and multiple sclerosis, respectively. Still other proteinsadministered are recombinant human deoxyribonuclease I(rhDNase-PULMOZYME®), an enzyme which selectively cleaves DNA used toimprove pulmonary function in patients with cystic fibrosis; recombinantthyroid stimulating hormone (THYROGEN®) developed for use in thyroidcancer patients who have had near-total or total thyroidectomy, and whomust therefore take thyroid hormones; recombinant G-CSF (NEUPOGEN®) fortreating neutropenia from chemotherapy, and digestive enzymes inindividuals with pancreatitis. Another significant area of proteintherapy is in the treatment of infectious diseases and cancer withantibodies, which have a highly specific, well-defined active site.Antibody therapeutic products include RESPIRGRAM® for respiratorysyncitial virus, HERCEPTIN®, for breast cancer; REMICAID® and HUMIRA®,for arthritis and inflammatory diseases, and others. ASSCs forantibodies are well known, and either the target antigen or astructurally related analog (e.g., a modified form of the active targetor a mimetic) can be employed. See Table 2 below for a list of proteinscurrently on the market or being evaluated in clinical trials for use asprotein therapy.

TABLE 2 Replacement Proteins Administered in Associated DisordersProtein Trade name Therapeutic function Development phase (rhuMAb-VEGF)Dynepo ™ anemia associated with Phase III renal disease α-L-iduronidaseAldurazyme ™ mucopolysaccharidosis-I Commercially available alronidaserDNA insulin diabetes Phase III alteplase, Activase ® acute myocardialCommercially infarction; acute massive available pulmonary embolism;ischemic stroke within 3 to 5 hours of symptom onset darbepoetin alfaAranesp ™ anemia Commercially available Deoxyribonuclease Pulmozymecystic fibrosis Commercially I available drotrecogin alfa Xigris ™severe sepsis Commercially (activated protein available C) efalizumabRaptiva ® moderate to severe Commercially psoriasis availableerythropoietin EPOGEN ® anemia Commercially available erythropoietinPROCRIT ® anemia Commercially available etanercept Enbrel ® rheumatoidCommercially arthritis; psoriatic arthritis available factor IXBeneFIX ™ hemophilia B Commercially available follicle-stimulatingFollistim ® infertility Commercially hormone available G-CSF Neupogenneutropenia resulted from Commercially Chemotherapy availableglucocerebrosidase Cerezyme ™ Gaucher's disease Commercially availableGM-CSF KGF mucositis Phase III (Repifermin) completed Growth hormoneBioTropin ™ growth hormone deficiency Commercially in children availableheat shock protein Leukine ® mucositis and melanoma Commerciallyavailable Insulin Humalog ® diabetes Commercially available interferonActimmune ® idiopathic pulmonary Commercially fibrosis availableinterferon alfa Enbrel ® ankylosing spondylitis, Commercially(enterecept) psoriasis available interferon alfa-2a, Roferon ®-A hairycell Commercially leukemia; Kaposi's available sarcoma; chronicrecombinant myelogenous leukemia; hepatitis C interferon alfa-n3Actimmune ® systemic fungal infections Commercially available interferonalfa-n3 Alferon N genital warts Commercially available interferonbeta-1a Avonex ® relapsing multiple Commercially sclerosis availableinterferon beta-1a Pegasys ® chronic hepatitis C Commercially availableinterferon beta-1b Betaseron ® relapsing, remitting Commerciallymultiple sclerosis available interferon beta-1b Rebif ® chronichepatitis C Commercially available interferon gamma Actimmune ® chronicgranulomatous Commercially 1b disease; osteopetrosis availableagalsidase beta Fabrazyme ™ Fabry disease Commercially availableinterleukin-2 Proleukin ® renal cell carcinoma; Commercially metastaticmelanoma available keratinocyte Avastin ™ colorectal cancer Phase IIIgrowth factor completed lepirudin Refludan ™ heparin-inducedCommercially (anticoagulant) thrombocytopenia type II availableomalizumab Xolair ® allergy-related asthma Commercially availablerasburicase Elitek ® hyperuricemia, Commercially available reteplase(tissue Retavase ® acute myocardial infarction Commercially plasminogenavailable factor) thyroid Thyrogen ® thyroid cancer Commerciallystimulating available hormone TNF-alpha Oncophage ® colorectal, renalcell Phase III cancer, melanoma trastuzumab Herceptin ® HER2overexpressing Commercially metastatic breast cancer available

Treatment of Protein Deficiencies and Other Disorders

As mentioned briefly above, gene therapy, protein replacement therapy,and small molecule inhibitor therapy have been developed as therapeuticstrategies for the treatment of genetic disorders resulting from proteindeficiencies and for disorders that benefit from administration ofreplacement proteins.

Protein replacement therapy increases the amount of protein byexogenously introducing wild-type or biologically functional protein byway of infusion. This therapy has been developed for many geneticdisorders including Gaucher disease, and Fabry disease, as referencedabove. The wild-type enzyme is purified from a recombinant cellularexpression system (e.g., mammalian cells or insect cells-see U.S. Pat.No. 5,580,757 to Desnick et al.; U.S. Pat. Nos. 6,395,884 and 6,458,574to Selden et al.; U.S. Pat. No. 6,461,609 to Calhoun et al.; U.S. Pat.No. 6,210,666 to Miyamura et al.; U.S. Pat. No. 6,083,725 to Selden etal.; U.S. Pat. No. 6,451,600 to Rasmussen et al.; U.S. Pat. No.5,236,838 to Rasmussen et al.; and U.S. Pat. No. 5,879,680 to Ginns etal.), human placenta, or animal milk (see U.S. Pat. No. 6,188,045 toReuser et al.). After the infusion, the exogenous enzyme is expected tobe taken up by tissues through non-specific or receptor-specificmechanism. In general, the uptake efficiency is not high, and thecirculation time of the exogenous protein is short (Ioannu et al., Am.J. Hum. Genet. 2001; 68: 14-25). In addition, the exogenous protein isunstable and subject to rapid intracellular degradation.

In addition to protein replacement and gene therapy, small moleculetherapy using enzyme inhibitors has been described for the treatment ofthe LSD's, namely small molecule inhibitors useful for substratedeprivation of the precursors of the deficient enzyme, referenced above.Small molecule inhibitors have been described for the treatment of LSD'sincluding Fabry disease, Gaucher disease, Pompe disease, Tay Sachsdisease, Sandhoff disease, and G_(M2) gangliosidoses (see U.S. Pat. Nos.5,472,969, 5,580,884, 5,798,366, and 5,801,185 to Platt et al.).

Co-Therapy Using ASSC's and Protein Replacement

The present invention increases the effectiveness of protein replacementtherapy by increasing the stability of the purified protein in vitro ina formulation or composition, and in vivo by co-administration of anASSC for the protein. Screening for an appropriate ASSC for the targetprotein can be achieved using ordinary methods in the art, for example,as described in U.S. patent application Ser. No. 10/377,179, filed Feb.28, 2003, which is incorporated herein by reference.

Replacement Protein Production

Disorders that can be treated using the method of the present inventioninclude but are not limited to LSD's, glucose-6-phosophate dehydrogenasedeficiency, hereditary emphysema, familial hypercholesterolemia,familial hypertrophic cardiomyopathy, phenylketonuria, anemia, hepatitisB and multiple sclerosis.

The replacement proteins useful for the methods of the present inventioncan be isolated and purified using ordinary molecular biology,microbiology, and recombinant DNA techniques within the skill of theart. For example, nucleic acids encoding the replacement protein can beisolated using recombinant DNA expression as described in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNACloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization [B. D. Hames & S. J. ÊHiggins eds. (1985)]; TranscriptionAnd Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal CellCulture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRLPress, (1986)]; B. ÊPerbal, A Practical Guide To Molecular Cloning(1984); F. M. Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, Inc. (1994). The nucleic acid encoding theprotein may be full-length or truncated, as long as the gene encodes abiologically active protein. For example, a biologically active,truncated form of α-Gal A, the defective enzyme associated with Fabrydisease, has been described in U.S. Pat. No. 6,210,666 to Miyamura etal.

The identified and isolated gene encoding the target protein can then beinserted into an appropriate cloning vector. A large number ofvector-host systems known in the art may be used. Possible vectorsinclude, but are not limited to, plasmids or modified viruses, but thevector system must be compatible with the host cell used. Examples ofvectors include, but are not limited to, E. coli, bacteriophages such aslambda derivatives, or plasmids such as pBR322 derivatives or pUCplasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. Theinsertion into a cloning vector can, for example, be accomplished byligating the DNA fragment into a cloning vector which has complementarycohesive termini. However, if the complementary restriction sites usedto fragment the DNA are not present in the cloning vector, the ends ofthe DNA molecules may be enzymatically modified. Alternatively, any sitedesired may be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. Production of the recombinant protein can: bemaximized by genetic manipulations such as including a signal peptide atthe N terminus to facilitate secretion or a 3′ untranslated sequencecontaining a polyadenylation site.

In a preferred embodiment, the constructs used to transduce host cellsare viral-derived vectors, including but not limited to adenoviruses,adeno-associated viruses, herpes virus, mumps virus, poliovirus,retroviruses, Sindbis virus and vaccinia viruses.

Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated. Preferably, the clonedgene is contained on a shuttle vector plasmid, which provides forexpansion in a cloning cell, e.g., E. coli, and facile purification forsubsequent insertion into an appropriate expression cell line, if suchis desired.

Potential host-vector systems include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.Different host cells have characteristic and specific mechanisms for thetranslational and post-translational processing and modification (e.g.,glycosylation, cleavage [e.g., of signal sequence]) of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed,such as glycosylation, sialyation and phosphorylation. For example,expression in a bacterial system can be used to produce annonglycosylated core protein product. However, protein expressed inbacteria may not be properly folded. Expression in yeast can produce aglycosylated product. Expression in eukaryotic cells can increase thelikelihood of “native” glycosylation and folding of a heterologousprotein. Moreover, expression in mammalian cells can provide a tool forreconstituting, or constituting, protein. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent. The expression efficiencycan be increased by use of a specific chaperone, as described in U.S.Pat. No. 6,274,597, and related family members disclosed above.

Purification of recombinantly expressed protein can be achieved usingmethods known in the art such as by ammonium sulfate precipitation,column chromatography containing hydrophobic interaction resins, cationexchange resins, anion exchange resins, and chromatofocusing resins.Alternatively, imunoaffinity chromatography can be used to purify therecombinant protein using an appropriate polyclonal or monoclonalantibody that binds specifically to the protein, or to a tag that isfused to the recombinant protein. In a preferred embodiment, the purityof the recombinant protein used for the method of the present inventionwith be at least 95%, preferably 97% and most preferably, greater than98%.

Replacement Protein Administration

Numerous methods can be employed to achieve uptake and targeting of thereplacement protein by the cells. Peptide sequences have been identifiedthat mediate membrane transport, and accordingly provide for delivery ofpolypeptides to the cytoplasm. For example, such peptides can be derivedfrom the Antennapedia homeodomain helix 3 to generate membrane transportvectors, such as penetratin (PCT Publication WO 00/29427; see alsoFischer et al., J. Pept. Res. 2000; 55:163-72; DeRossi et al., Trends inCell Biol. 1998; 8:84-7; Brugidou et al., Biochem. Biophys. Res. Comm.1995; 214:685-93), the VP22 protein from herpes simplex virus (Phelan etal., Nat. Biotechnol. 1998; 16:440-3), and the HIV TAT trascriptionalactivator. Protein transduction domains, including the Antennapediadomain and the HIV TAT domain (see Vives et al., J. Biol. Chem. 1997;272:16010-17), possess a characteristic positive charge, which led tothe development of cationic 12-mer peptides that can be used to transfertherapeutic proteins and DNA into cells (Mi et al., Mol. Therapy 2000;2:339-47). The above-mentioned protein transduction domains arecovalently linked to the target protein, either by chemical covalentcross-linking or generation as a fusion protein. Further, anon-covalent, synthetic protein transduction domain has been recentlydeveloped by Active Motif Inc. (Carlsbad, Calif.). This domainassociates with the target protein through hydrophobic interactions, andadvantageously dissociates from the protein once inside the cell (Morriset al., Nat. Biotechnol. 2001; 19:1173-6). In addition, lipid carriershave recently been shown to deliver proteins into cells in addition toan established use for delivering naked DNA (Zelphati et al., J. Biol.Chem. 2001; 276:35103-10). For an overview of protein translocationtechniques see Bonetta, The Scientist 2002; 16(7):38.

In specific embodiments, the replacement proteins used in the method ofthe present invention are enzymes associated with lysosomal storagedisorders (see Table 1). Sequences of nucleic acids encoding wild-typeversions of such enzymes can be found in the literature or in publicdatabases such as GenBank, e.g., X14448 for α-Gal A (AGA), J03059 forhuman glucocerebrosidase (GCB), M74715 for human α-L-iduronidase (IDUA),M34424 for human acid α-glucosidase (GAA), AF011889 for human iduronate2-sulfatase (IDS), and M59916 for human acid sphingomyelinase (ASM).

Enzyme Replacement in LSDs.

Several replacement enzymes for LSDs are currently available in Europeand the U.S. These include Cerezyme®, recombinant form ofglucerebrosidase for the treatment of Gaucher disease; Fabrazyme®,recombinant form of alpha galactosidase A; Aldurazyme™, a recombinantenzyme for the treatment of MPS1, all from Genzyme Corp. and recombinantalpha glucosidase for patients with Pompe disease (Van den Hout et al.,Lancet 2000; 56:397-8).

Active Site-Specific Chaperones

ASSC's contemplated by the present invention include but are not limitedto small molecules (e.g., organic or inorganic molecules which are lessthan about 2 kD in molecular weight, are more preferably less than about1 kD in molecular weight), including substrate or binding partnermimetics; small ligand-derived peptides or mimetics thereof; nucleicacids such as DNA, RNA; antibodies, including Fv and single chainantibodies, and Fab fragments; macromolecules (e.g., molecules greaterthan about 2 kD in molecular weight) and members of libraries derived bycombinatorial chemistry, such as molecular libraries of D- and/orL-configuration amino acids; phosphopeptides, such as members of randomor partially degenerate, directed phosphopeptide libraries (see, e.g.,Songyang et al., Cell 1993; 72:767-778).

Synthetic libraries (Needels et al., Proc. Natl. Acad. Sci. USA 1993;90:10700-4; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 1993;90:10922-10926; Lam et al., PCT Publication No. WO 92/00252; Kocis etal., PCT Publication No. WO 94/28028) provide a source of potentialASSC's according to the present invention. Synthetic compound librariesare commercially available from Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemicallibrary is available from Aldrich (Milwaukee, Wis.). Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available from e.g. Pan Laboratories (Bothell,Wash.) or MycoSearch (NC), or are readily producible. Additionally,natural and synthetically produced libraries and compounds are readilymodified through Res. 1986; 155:119-29.

In a preferred embodiment, ASSC's useful for the present invention areinhibitors of lysosomal enzymes and include glucose and galactoseimino-sugar derivatives as described in Asano et al., J. Med. Chem 1994;37:3701-06; Dale et al., Biochemistry. 1985; 24:3530-39; Goldman et al.,J. Nat. Prod. 1996; 59:1137-42; Legler et al, Carbohydrate Res. 1986;155:119-29. Such derivatives include but are not limited those compoundlisted in Table 1. Some of these compounds can be purchased fromcommercial sources such as Toronto Research Chemicals, Inc. (North York,On. Canada) and Sigma.

In a preferred embodiment, ASSC's useful for the present invention areactivators of cystic fibrosis transmembrane conductance regulator (CFTR)which include benzo(c)quinolizinium compounds as described in Dormer etal., J. Cell Sci. 2001; 114: 4073-81; and Ma et al., J. Biol. Chem.2002; 277: 37235-41.

In another preferred embodiment, ASSC's useful for the present inventionare ligands of G protein-coupled receptors, such as 0 opioid receptor,V2 vasopressin receptor, and photopigment rhodopsin, as described inPetaja-Repo et al., EMBO J 2002; 21: 1628-37; Morello et al., J. Clin.Invest. 2000; 105: 887-95; Saliba et al., J. Cell Sci. 2002; 115:2907-18.

In another preferred embodiment, ASSC's useful for the present inventionare compounds that stabilize the DNA binding domain of p53, as describedin Foster et al., Science 1999; 286: 2507-10; Friedler et al., PNAS2002; 99: 937-42.

In yet another preferred embodiment, ASSC's useful for the presentinvention are blockers of ion channel proteins, such as HERG potassiumchannel in human Long QT syndrome, pancereatic ATP-sensitive potassium(K_(ATP)) channel in familial hyperinsulinism, as described in Zhou etal., J Biol. Chem. 1999; 274: 31123-26; Taschenberger et al., J. Biol.Chem. 2002; 277: 17139-46.

Formulations

In one embodiment, the ASSC and replacement protein are formulated in asingle composition. Such a composition enhances stability of the proteinduring storage and in vivo administration, thereby increasingtherapeutic efficacy. The formulation is preferably suitable forparenteral administration, including intravenous subcutaneous, andintraperitoneal, however, formulations suitable for other routes ofadministration such as oral, intranasal, or transdermal are alsocontemplated.

In another embodiment, the replacement protein and the ASSC's areformulated in separate compositions. In this embodiment, the chaperoneand the replacement protein may be administered according to the sameroute, e.g., intravenous infusion, or different routes, e.g.,intravenous infusion for the replacement protein, and oraladministration for the ASSC. The pharmaceutical formulations suitablefor injectable use include sterile aqueous solutions (where watersoluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. In all cases,the form must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Thepreventions of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, benzyl alcohol, sorbic acid, and the like. Inmany cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonosterate and gelatin.

Sterile injectable solutions are prepared by incorporating the purifiedprotein and ASSC in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter or terminal sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

Preferably the formulation contains an excipient. Pharmaceuticallyacceptable excipients which may be included in the formulation arebuffers such as citrate buffer, phosphate buffer, acetate buffer, andbicarbonate buffer, amino acids, urea, alcohols, ascorbic acid,phospholipids; proteins, such as serum albumin, collagen, and gelatin;salts such as EDTA or EGTA, and sodium chloride; liposomes;polyvinylpyrollidone; sugars, such as dextran, mannitol, sorbitol, andglycerol; propylene glycol and polyethylene glycol (e.g., PEG-4000,PEG-6000); glycerol; glycine or other amino acids; and lipids. Buffersystems for use with the formulations include citrate; acetate;bicarbonate; and phosphate buffers. Phosphate buffer is a preferredembodiment.

The formulation also preferably contains a non-ionic detergent.Preferred non-ionic detergents include Polysorbate 20, Polysorbate 80,Triton X-100, Triton X-114, Nonidet P-40, Octyl α-glucoside, Octylβ-glucoside, Brij 35, Pluronic, and Tween 20.

For lyophilization of protein and chaperone preparations, the proteinconcentration can be 0.1-10 mg/mL. Bulking agents, such as glycine,mannitol, albumin, and dextran, can be added to the lyophilizationmixture. In addition, possible cryoprotectants, such as disaccharides,amino acids, and PEG, can be added to the lyophilization mixture. Any ofthe buffers, excipients, and detergents listed above, can also be added.

Formulations for inhalation administration may contain lactose or otherexcipients, or may be aqueous solutions which may containpolyoxyethylene-9-lauryl ether, glycocholate or deoxycocholate. Apreferred inhalation aerosol is characterized by having particles ofsmall mass density and large size. Particles with mass densities lessthan 0.4 gram per cubic centimeter and mean diameters exceeding 5 μmefficiently deliver inhaled therapeutics into the systemic circulation.Such particles are inspired deep into the lungs and escape the lungs'natural clearance mechanisms until the inhaled particles deliver theirtherapeutic payload. (Edwards et al., Science 1997; 276: 1868-1872).Replacement protein preparations of the present invention can beadministered in aerosolized form, for example by using methods ofpreparation and formulations as described in, U.S. Pat. Nos. 5,654,007,5,780,014, and 5,814,607, each incorporated herein by reference.Formulation for intranasal administration may include oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally.

Formulations for topical administration to the skin surface may beprepared by dispersing the composition with a dermatological acceptablecarrier such as a lotion, cream, ointment, or soap. Particularly usefulare carriers capable of forming a film or layer over the skin tolocalize application and inhibit removal. For topical administration tointernal tissue surfaces, the composition may be dispersed in a liquidtissue adhesive or other substance known to enhance adsorption to atissue surface. Alternatively, tissue-coating solutions, such aspectin-containing formulations may be used.

In preferred embodiments, the formulations of the invention are suppliedin either liquid or powdered formulations in devices which convenientlyadminister a predetermined dose of the preparation; examples of suchdevices include a needle-less injector for either subcutaneous orintramuscular injection, and a metered aerosol delivery device. In otherinstances, the preparation may be supplied in a form suitable forsustained release, such as in a patch or dressing to be applied to theskin for transdermal administration, or via erodable devices fortransmucosal administration. In instances where the formulation, e.g.,the ASSC is orally administered in tablet or capsule form, thepreparation might be supplied in a bottle with a removable cover or asblister patches.

In Vitro Stability.

Ensuring the stability of a pharmaceutical formulation during its shelflife is a major challenge. Prior to development of a proteinpharmaceutical, inherent or latent instabilities within the activeingredients must be explored and addressed. Instability of protein andpeptide therapeutics is classified as chemical instability or physicalinstability. Examples of chemical instability are hydrolysis, oxidationand deamidation. Examples of physical instability are aggregation,precipitation and adsorption to surfaces. In addition, a protein may besubjected to stresses such as pH, temperature, shear stress, freeze/thawstress and combinations of these stresses.

One of the most prevalent formulation problems is product aggregation,resulting in a loss in bioactivity. The addition of excipients may slowthe process but may not completely prevent it. Activity losses may ormay not be detected by physical assays and are only evident in bioassaysor potency assays with large (sometimes 15-20%) coefficients ofvariation, making it difficult to determine actual losses.

ASSC have been shown to enhance enzyme activity by preventingdegradation of enzymes and aggregation of enzyme proteins (Fan et al.,Nat. Med. 1999; 5: 112-5; FIG. 1). In the embodiment where the ASSC andthe replacement protein are in the same composition, the formulatedcompositions of the invention may be provided in containers suitable formaintaining sterility, and importantly, protecting the activity of thereplacement protein during proper distribution and storage. In additionto stabilizing the administered protein in vivo, the ASSC reversiblybinds to and stabilizes the conformation of the replacement protein invitro, thereby preventing aggregation and degradation, and extending theshelf-life of the formulation. Analysis of the ASSC/replacement proteininteraction may be evaluated using techniques well-known in the art,such as, for example, differential scanning calorimetry, or circulardichroism.

For example, where an aqueous injectable formulation of the compositionis supplied in a stoppered vial suitable for withdrawal of the contentsusing a needle and syringe, the presence of an ASSC inhibits aggregationof the replacement protein. The vial could be for either single use ormultiple uses. The formulation can also be supplied as a prefilledsyringe. In another embodiment, the formulation is in a dry orlyophilized state, which would require reconstitution with a standard ora supplied, physiological diluent to a liquid state. In this instance,the presence of an ASSC would stabilize the replacement protein duringand post-reconstitution to prevent aggregation. In the embodiment wherethe formulation is a liquid for intravenous administration, such as in asterile bag for connection to an intravenous administration line orcatheter, the presence of an ASSC would confer the same benefit.

In addition to stabilizing the replacement protein to be administered,the presence of an ASSC may enable the pharmaceutical formulation to bestored at a neutral pH of about 7.0-7.5. This will confer a benefit toproteins that normally must be stored at a lower pH to preservestability. For example, lysosomal enzymes, such as those listed in Table1, retain a stable conformation at a low pH (e.g., 5.0 or lower).However, extended storage of the replacement enzyme at a low pH mayexpedite degradation of the enzyme and/or formulation.

Separate Formulations.

Where the replacement enzyme and ASSC are in separate formulations, theASSC can be in a form suitable for any route of administration,including all of the forms described above, e.g., as sterile aqueoussolution or in a dry lyophilized powder to be added to the formulationof the replacement protein during or immediately after reconstitution toprevent aggregation in vitro prior to administration. Alternatively, theASSC can be formulated for oral administration in the form of tablets orcapsules prepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell known in the art. Liquid preparations for oral administration maytake the form of, for example, solutions, syrups or suspensions, or theymay be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

Administration

The route of administration may be oral or parenteral, includingintravenous, subcutaneous, intra-arterial, intraperitoneal, ophthalmic,intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral,intradermal, intracranial, intraspinal, intraventricular, intrathecal,intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal,transdermal, or via inhalation.

Administration of the above-described parenteral formulations may be byperiodic injections of a bolus of the preparation, or may beadministered by intravenous or intraperitoneal administration from areservoir which is external (e.g., an i.v. bag) or internal (e.g., abioerodable implant, a bioartificial organ, or a population of implantedcells that produce the replacement protein). See, e.g., U.S. Pat. Nos.4,407,957 and 5,798,113, each incorporated herein by reference.Intrapulmonary delivery methods and apparatus are described, forexample, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aeorosolizer, electroporation, and transdermalpatch. Needle-less injector devices are described in U.S. Pat. Nos.5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the formulationsdescribed above can administered in these methods.

Subcutaneous injections the replacement protein and/or ASSC have theadvantages allowing self-administration, while also resulting in aprolonged plasma half-life as compared to intravenous administration.Furthermore, a variety of devices designed for patient convenience, suchas refillable injection pens and needle-less injection devices, may beused with the formulations of the present invention as discussed herein.

Timing.

When the replacement protein and ASSC are in separate formulations,administration may be simultaneous, or the ASSC may be administeredprior to, or after the replacement protein. For example, where thereplacement protein is administered intravenously, the ASSC may beadministered during a period from 0 h to 6 h later. Alternatively, thechaperone may be administered from 0 to 6 h prior to the protein.

In a preferred embodiment, where the ASSC and replacement protein areadministered separately, and where the ASSC has a short circulatinghalf-life (e.g., small molecule), the ASSC may be orally administeredcontinuously, such as daily, in order to maintain a constant level inthe circulation. Such constant level will be one that has beendetermined to be non-toxic to the patient, and optimal regardinginteraction with a target replacement protein during the time ofadministration to confer a non-inhibitory, therapeutic effect.

In another embodiment, the ASSC is administered during the time periodrequired for turnover of the replacement protein (which will be extendedby administration of the ASSC).

Regardless of the timing, the administration must be such that theconcentrations of the protein and ASSC must be such that the chaperonestabilizes, but does not prevent or inhibit the protein's activity invivo. This also applies where the replacement protein and ASSC areadministered in the same formulation.

In Vivo Stability.

As described above for the in vitro formulations, the presence of anASSC for the replacement protein will have the benefit of prolonging inplasma the half-life, thereby maintaining effective replacement proteinlevels over longer time periods, resulting in increased exposure ofclinically affected tissues to the replacement protein and, thus,increased uptake of protein into the tissues. This confers suchbeneficial effects to the patient as enhanced relief, reduction in thefrequency, and/or reduction in the amount administered. This will alsoreduce the cost of treatment.

In addition to stabilizing wild-type replacement proteins, the ASSC willalso stabilize and enhance expression of endogenous mutant proteins thatare deficient as a result of mutations that prevent proper folding andprocessing in the ER, as in conformational disorders such as the LSDs.

Dosages

The amount of ASSC effective to stabilize the administered protein andendogenous mutant protein can be determined on a case-by-case basis,depending on the protein and corresponding ASSC, by those skilled in theart. Pharmacokinetics and pharmacodynamics such as half-life (t₁₋₂),peak plasma concentration (c_(max)), time to peak plasma concentration(t_(max)), exposure as measured by area under the curve (AUC); andtissue distribution for both the replacement protein and the ASSC, aswell as data for ASSC-replacement protein binding (affinity constants,association and dissociation constants, and valency), can be obtainedusing ordinary methods known in the art to determine compatible amountsrequired to stabilize the replacement protein, without inhibiting itsactivity, and thus confer a therapeutic effect.

Data obtained from cell culture assay or animal studies may be used toformulate a therapeutic dosage range for use in humans and non-humananimals. The dosage of compounds used in therapeutic methods of thepresent invention preferably lie within a range of circulatingconcentrations that includes the ED₅₀ concentration (effective for 50%of the tested population) but with little or no toxicity. The particulardosage used in any treatment may vary within this range, depending uponfactors such as the particular dosage form employed, the route ofadministration utilized, the conditions of the individual (e.g.,patient), and so forth.

A therapeutically effective dose may be initially estimated from cellculture assays and formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀, The IC₅₀ concentration of acompound is the concentration that achieves a half-maximal inhibition ofsymptoms (e.g., as determined from the cell culture assays). Appropriatedosages for use in a particular individual, for example in humanpatients, may then be more accurately determined using such information.

Measures of compounds in plasma may be routinely measured in anindividual such as a patient by techniques such as high performanceliquid chromatography (HPLC) or gas chromatography.

Toxicity and therapeutic efficacy of the composition can be determinedby standard pharmaceutical procedures, for example in cell cultureassays or using experimental animals to determine the LD₅₀ and the ED₅₀.The parameters LD₅₀ and ED₅₀ are well known in the art, and refer to thedoses of a compound that is lethal to 50% of a population andtherapeutically effective in 50% of a population, respectively. The doseratio between toxic and therapeutic effects is referred to as thetherapeutic index and may be expressed as the ratio: LD₅₀/ED₅₀. ASSCsthat exhibit large therapeutic indices are preferred.

According to current methods, the concentration of replacement proteinis between 0.05-5.0 mg/kg of body weight, typically administered weeklyor biweekly. The protein can be administered at a dosage ranging from0.1 μg/kg to about 10 mg/kg, preferably from about 0.1 mg/kg to about 2mg/kg. For example, for the treatment of Fabry disease, the dose ofrecombinant α-Gal A administrated is typically between 0.1-0.3 mg/kg andis administered weekly or biweekly. Regularly repeated doses of theprotein are necessary over the life of the patient. Subcutaneousinjections maintain longer term systemic exposure to the drug. Thesubcutaneous dosage is preferably 0.1-5.0 mg of the α-Gal A per kg bodyweight biweekly or weekly. The α-Gal A is also administeredintravenously, e.g., in an intravenous bolus injection, in a slow pushintravenous injection, or by continuous intravenous injection.Continuous IV infusion (e.g., over 2-6 hours) allows the maintenance ofspecific levels in the blood.

The optimal concentrations of the ASSC will be determined according tothe amount required to stabilize the recombinant protein in vivo, intissue or circulation, without preventing its activity, bioavailabilityof the ASSC in tissue or in circulation, and metabolism of the ASSC intissue or in circulation. For example, where the ASSC is an enzymeinhibitor, the concentration of the inhibitor can be determined bycalculating the IC₅₀ value of the specific chaperone for the enzyme.Taking into consideration bioavailability and metabolism of thecompound, concentrations around the IC₅₀ value or slightly over the IC₅₀value can then be evaluated based on effects on enzyme activity, e.g.,the amount of inhibitor needed to increase the amount of enzyme activityor prolong enzyme activity of the administered enzyme. As an example,the IC₅₀ value of the compound deoxygalactonojiromycin (DGJ) for theα-Gal A enzyme is 0.04 μM, indicating that DGJ is a potent inhibitor.Accordingly, it is expected that the intracellular concentration ofα-Gal A would be much lower than that of the α-Gal A administered. SeeExamples below.

EXAMPLES

The present invention is further described by means of the examples,presented below. The use of such examples is illustrative only and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to any particular preferredembodiments described herein. Indeed, many modifications and variationsof the invention will be apparent to those skilled in the art uponreading this specification and can be made without departing from itsspirit and scope. The invention is therefore to be limited only by theterms of the appended claims along with the full scope of equivalents towhich the claims are entitled.

Example 1: In Vitro Stabilization of α-Gal A with ASSCs

Methods.

The wild type α-Gal A was purified from culture medium of Sf-9 cellsinfected with recombinant baculovirus carrying human wild type α-Gal AcDNA and the mutant α-Gal A was collected as homogenates of hearts oftransgenic mice overexpressing human mutant (R301Q) α-Gal A. The micewere treated with 0.5 mM DGJ as drinking water for one week prior to theexperiment. The mutant and wild type enzymes were pre-incubated with 0.1M citrate-phosphate buffer (pH 7.0) at 37° C. for the mutant enzyme and42° C. for the wild type enzyme, respectively, in the presence of DGJ ata concentration of 1 μM, 0.1 μM, 0.03 μM or no DGJ. The wild type andmutant (R301Q) α-Gal A were incubated for a period of time in theabsence or presence of DGJ (various concentrations), and the remainingenzyme activity was determined with 4-MU-α-Gal A as a substrate; afterdiluting the mixture with 5-volume of 0.1 M citrate buffer (pH 4.5).Enzyme activity is reported relative to the enzyme withoutpre-incubation.

Results.

As shown in FIG. 1, the mutant enzyme was not stable at neutral pH afterincubation at 37° C. for 20 min without incubation with DGJ (FIG. 1A).The wild type enzyme also lost significant enzyme activity at neutral pHat 42° C. without incubation with DGJ (FIG. 1B). The stability of bothenzymes can be improved by inclusion of DGJ at 1 μM concentration, i.e.,more than 80% of enzyme activity was remained in the reaction mixturefor 60 min. This indicates that the ASSC (DGJ) can serve as a stabilizerto prevent the denaturation/degradation of the mutant and wild typeenzymes.

Example 2: Intracellular Enhancement of Wild-Type α-Gal A with ASSCs

Methods.

Human wild type α-Gal A purified from insect cells transfected withrecombinant baculovirus or from recombinant CHO cells can be conjugatedto α-2-macroglobulin (α-2-M), according to the previous reference (Osadaet al., Biochem Biophys Res Commun. 1993; 142: 100-6). Since theconjugate of α-Gal A from coffee beans and α-2-M can be internalized bycultured fibroblasts derived from Fabry hemizygotes, the conjugate ofα-Gal A and α-2-M is expected to be internalized by the cells as well.Alternatively, the wild type α-Gal A can be added into the culturemedium of skin fibroblasts derived from Fabry patient with no residualenzyme activity as described in Blom et al., Am J Hum Gen. 2003; 72:23-31.

Results.

The half-life of the coffee bean α-Gal A is about 2 hr as describedpreviously (Osada et al., Biochem Biophys Res Commun. 1987; 143: 954-8).It is expected that the half-life of the α-Gal A/α-2-M conjugate orα-Gal A added into the culture medium can be extended by inclusion ofDGJ into the culture medium, since the DGJ has been shown to beeffective in stabilize the enzyme in vitro (FIG. 1). This will indicatethat the DGJ can prolong the exogenous α-Gal A taken up by the cellsintracellularly.

Example 3: Co-Administration of DGJ to Fabry Mice Treated by Infusion ofReplacement Enzyme

Enzyme replacement therapy for Fabry disease has been developed byGenzyme Corporation as described above. It is expected thatco-administration of DGJ to Fabry knock-out (KO) mice treated byinfusion of the replacement enzyme increases the stability, e.g.,half-life of the replacement enzyme in vivo, because the ASSC DGJstabilizes the enzyme and prevents degradation. DGJ is orallyadministered to the KO mice after infusion of the wild type α-Gal Aaccording to the protocol described previously (Ioannu et al., Am J HumGenet. 2001; 68:14-25). The α-Gal A activity in various tissuesincluding heart, kidney, spleen, liver, and lung as well as serum isdetermined over a period of time, and compared with those from thecontrol mice that do not receive DGJ, and mice that receive only DGJ butno enzyme. The extended time will indicate that co-administration ofASSC can improve the efficiency of enzyme replacement therapy.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, procedures, and the like arecited throughout this application, the disclosures of which areincorporated herein by reference in their entireties.

1-20. (canceled) 21: A stable pharmaceutical composition formulated forparenteral administration to a human and comprising a purifiedrecombinant human wild-type α-galactosidase A and1-deoxygalactonojirimycin in a pharmaceutically acceptable carrier. 22:The stable pharmaceutical composition according to claim 21, which isformulated for intravenous, subcutaneous, or intraperitonealadministration. 23: The stable pharmaceutical composition according toclaim 21, further comprising: a buffer selected from the groupconsisting of a citrate buffer, a phosphate buffer, an acetate buffer,and a bicarbonate buffer. 24: A method of treating Fabry disease, themethod comprising: administering the stable pharmaceutical compositionaccording to claim 21 to a human patient in need thereof.